The purpose of this proposal is to transform screening and diagnosis for prostate cancer (PrCa) by using new methods for photoacoustic imaging (PAI) coupled with novel near infrared (NIR) imaging agents that specifically target PrCa tissue. The targeted molecular imaging agents (TMIAs) are based on exogenous NIR fluorescent (NIRF) and quencher (NIRQ) dyes which will be used singly and in combination to specifically target PrCa tissue. The technology builds on recent advances made in PAI instrumentation and techniques using endogenous dyes for the PAI of PrCa in a new research collaboration between researchers at the Rochester Institute of Technology (RIT) and the University of Rochester Medical Center (URMC). There is a significant need to affordably provide confirmation for PSA (prostate specific antigen) screening, reliably guide needle biopsies and monitor therapy for PrCa. Our strategy incorporates new synthetic methods which we have developed at RIT for preparing single and dual modal imaging agents built on a peptide scaffold with features that facilitate their gentle conjugation to biomolecules including inhibitor, peptides, antibodies and aptamers. The synthesis of pre-formed NIRQ-NIRF dye pairs on the side chains of the peptide scaffold will be followed by conjugation to two different targeting motifs for the catalytic site of prostate specific membrane antigen (PSMA). These targeting motifs are: a small molecule inhibitor related to the small urea YC-27; and an RNA aptamer, A10-3.2. The membrane antigen, prostate specific membrane antigen (PSMA), is expressed on almost all prostate cancers and relatively few other tissues making it a highly specific target for imaging. Moreover it has already been successfully targeted in other PrCa imaging applications and used as a therapy target. Thus, it represents a well- established target for PrCa imaging that is ideal for the NIRF-NIRQ approach to enable specificity, sensitivity and increase depth of imaging by PAI. The binding efficacy to PrCa cells will be evaluated by in-vitro confocal fluorescence microscopy (CFM) using TMIAs containing NIRF dyes to ensure robustness of the targeting strategy. In-vitro evaluation of TMIAs in PAI will be performed at URMC under the guidance of imaging scientists with expertise in the application of in-vitro phantoms for PAI of PrCa. Key to this proposal is the involvement of four undergraduates and one graduate student from RIT in each year, two of whom will also conduct research at URMC. The collaboration highlights expertise in imaging agent synthesis and evaluation by CFM at RIT and the expertise in cancer biology and PAI at URMC. These provide a vibrant learning environment in cancer imaging research for undergraduates with the achievable goal of transforming screening and diagnosis of PrCa, and directing biopsies by PAI: a new, reliable and inexpensive imaging method.